It has long been recognized that parasitic micro-organisms possess the ability to infect animals thereby causing disease and often the death of the host. Pathogenic agents have been a leading cause of death throughout out history and continue to inflict immense suffering. Though the last hundred years have seen dramatic advances in the prevention and treatment of many infectious diseases, complicated host-parasite interactions still limit the universal effectiveness of therapeutic measures. Difficulties in countering the sophisticated invasive mechanisms displayed by many pathogenic vectors is evidenced by the resurgence of various diseases such as tuberculosis, as well as the appearance of numerous drug resistant strains of bacteria and viruses.
Among those pathogenic agents of major epidemiological concern, intracellular organisms have proven to be particularly intractable in the face of therapeutic or prophylactic measures. Intracellular organisms, including bacteria of the genus Mycobacterium and the genus Legionella, complete all or part of their life cycle within the cells of the infected host organism rather than extracellularly. Around the world, intracellular bacteria are responsible for millions of deaths each year and untold suffering. Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of death from infectious disease worldwide, with 10 million new cases and 2.9 million deaths every year. In addition, intracellular bacteria are responsible for millions of cases of leprosy. Other debilitating diseases transmitted by intracellular agents include cutaneous and visceral leishmaniasis, American trypanosomiasis (Chagas' disease), listeriosis, toxoplasmosis, histoplasmosis, trachoma, psittacosis, Q-fever, and legionellosis including Legionnaires' disease. At this time, treatment and prevention of these diseases is suboptimal. In many cases, relatively little can be done to prevent debilitating infections in susceptible individuals exposed to these organisms.
Due to this inability to effectively protect population from tuberculosis and the inherent human morbidity and mortality caused by tuberculosis, this is one of the most important diseases confronting mankind. More specifically, human pulmonary tuberculosis primarily caused by M. tuberculosis is a major cause of death in developing countries. By concealing itself within the cells primarily responsible for the detection of foreign elements and subsequent activation of the immune system, M. tuberculosis is relatively successful in evading the normal defenses of the host organism. As a result, by surviving inside macrophages and monocytes, M. tuberculosis may produce a chronic intracellular infection. These same pathogenic characteristics have heretofore prevented the development of effective prophylaxis or chemotherapeutic methods and agents against tubercular infections.
Those skilled in the art will appreciate that the discussion of M. tuberculosis is in no way intended to limit the scope of the present invention to the treatment of tubercular infections. On the contrary, this invention may be used to advantageously provide safe and effective prophylactic and chemotherapeutic methods and agents against any susceptible disease caused by intracellular pathogenic agents.
Currently it is believed that approximately one third of the world's population is infected by M. tuberculosis resulting in millions of cases of pulmonary tuberculosis annually. While this disease is a particularly acute health problem in the developing countries of Latin America, Africa, and Asia, it is also becoming more prevalent in the first world. In the United States specific populations are at increased risk, especially urban poor, immunocompromised individuals and immigrants from areas of high disease prevalence. Largely due to the AIDS epidemic the incidence of tuberculosis is presently increasing in developed countries, often in the form of multi-drug resistant M. tuberculosis.
Recently, tuberculosis resistance to one or more drugs was reported in 36 of the 50 United States. In New York City, one-third of all cases tested in 1991 were resistant to one or more major drugs. Though non-resistant tuberculosis can be cured with a long course of antibiotics, the outlook regarding drug resistant strains is bleak. Patients infected with strains resistant to two or more major antibiotics have a fatality rate of around 50%. Accordingly, a safe and effective treatment against such varieties of M. tuberculosis is sorely needed.
Initial infections of M. tuberculosis almost always occur through the inhalation of aerosolized particles, since the pathogen can remain viable for weeks or months in moist or dry sputum. Although the primary site of the infection is in the lungs, the organism can also cause infection of the bones, spleen, meninges and skin. Depending on the virulence of the particular strain and the resistance of the host, the infection and corresponding damage to the tissue may be minor or extensive. In the case of humans, the initial infection is controlled in the majority of individuals exposed to virulent strains of the bacteria. The development of acquired immunity following the initial challenge reduces bacterial proliferation thereby allowing lesions to heal and leaving the subject largely asymptomatic. However, approximately 10% of such individuals may reactivate the disease at some time in their lifetime, often many years after the initial infection. Reactivation disease can be debilitating, fatal, and contagious.
When M. tuberculosis is not controlled by the infected subject, it often results in the extensive degradation of lung tissue. In susceptible individuals lesions are usually formed in the lung as the tubercle bacilli reproduce within alveolar or pulmonary macrophages. As the organisms multiply, they may spread through the lymphatic system to distal lymph nodes and through the blood stream to the lung apices, bone marrow, kidney, and meninges surrounding the brain. Primarily as the result of cell-mediated hypersensitivity responses, characteristic granulomatous lesions or tubercles are produced in proportion to the severity of the infection. These lesions consist of epithelioid cells bordered by monocytes, lymphocytes and fibroblasts. In most instances a lesion or tubercle eventually becomes necrotic and undergoes caseation.
While M. tuberculosis is a significant pathogen, other species of the genus Mycobacterium also cause disease in animals including man and are clearly within the scope of the present invention. For example, M. bovis is closely related to M. tuberculosis and is responsible for tubercular infections in domestic animals such as cattle, pigs, sheep, horses, dogs and cats. Further, M. bovis may infect humans via the intestinal tract, typically from the ingestion of raw milk. The localized intestinal infection eventually spreads to the respiratory tract and is followed shortly by the classic symptoms of tuberculosis. M. avium can cause serious infection in immunocompromised people including patients with AIDS. Another important pathogenic vector of the genus Mycobacterium is M. leprae which causes millions of cases of the ancient disease, leprosy. Other species of this genus which cause disease in animals and man include M. kansasii, M. fortuitum, M. marinum, M. chelonei, M. africanum, M. ulcerans, M. microti and M. scrofulaceum. The pathogenic mycobacterial species frequently exhibit a high degree of homology in their respective DNA and corresponding protein and enzyme sequences and some species, such as M. tuberculosis and M. bovis, are highly related.
Any animal or human infected with a pathogen and, in particular, an intracellular organism, presents a difficult challenge to the host immune system. While many infectious agents may be effectively controlled by the humoral response and corresponding production of protective antibodies, these mechanisms are primarily effective only against those pathogens located in the body's extracellular fluid. In particular, opsonizing antibodies bind to extracellular foreign agents thereby rendering them susceptible to phagocytosis and subsequent intracellular killing. Yet this is not the case for other pathogens. For example, previous studies have indicated that the humoral immune response does not appear to play a significant protective role against infections by intracellular bacteria such as M. tuberculosis.
Antibody mediated defenses seemingly do not prevent the initial infection of such intracellular pathogens and are ineffectual once the bacteria are sequestered within the cells of the host. As water soluble proteins, antibodies can permeate the extracellular fluid and blood, but have difficulty migrating across the lipid membranes of cells to the sequestered intracellular bacteria. Further, the production of opsonizing antibodies against bacterial surface structures may actually assist intracellular pathogens in entering the host cell.
Unlike most infectious bacteria, mycobacteria, including M. tuberculosis, tend to proliferate in vacuoles which are substantially sealed off from the rest of the infected cell by a membrane. Phagocytes naturally form these protective vacuoles making them particularly susceptible to infection by this class of pathogen.
The problems intracellular pathogens pose for the infected host's immune system also constitute a special challenge to the medical communities' development of effective prophylactic and chemotherapeutic regimes. At the present time there are few effective treatments against intracellular pathogens.
In this regard, extracellular products or their immunogenic analogs have been used to stimulate prophylactic or protective immunity against intracellular pathogens. For example, U.S. Pat. No. 5,108,745, issued Apr. 28, 1992, to Marcus A. Horwitz, discloses vaccines and methods of producing protective immunity against Legionella pneumophila and Mycobacterium tuberculosis as well as other intracellular pathogens. Unlike traditional vaccines which rely on the use of attenuated pathogens or non-infectious components of the pathogens themselves to stimulate a protective immune response, these prior art vaccines are broadly based on the use of extracellular products. Originally derived from proteinaceous and other compounds released extracellularly by the pathogenic bacteria into broth culture in vitro and released extracellularly by bacteria within infected host cells in vivo, these vaccines are based on the identification of extracellular products or their analogs which stimulate a strong immune response against the target pathogen in a mammalian host.
More specifically, these prior art candidate extracellular products were screened by determining their ability to provoke either a strong lymphocyte proliferative response or a cutaneous delayed-type hypersensitivity response in mammals which were immune to the pathogen of interest. Following the growth and harvesting of the bacteria, by virtue of their physical abundance, the principal extracellular products were separated from intrabacterial and other components through centrifugation and filtration. If desired, the resultant bulk filtrate could be then subjected to fractionation using ammonium sulfate precipitation with subsequent dialysis to give a mixture of extracellular products, commonly termed EP. Solubilized extracellular products in the dialyzed fractions were then purified to substantial homogeneity using suitable chromatographic techniques as known in the art.
These exemplary procedures resulted in the identification of dozens of compounds and in the subsequent production of fourteen individual proteinaceous major extracellular products, referred to as the majorly abundant extracellular products of M. tuberculosis, having molecular weights ranging from 110 kilo Daltons (KD) to 12 KD. Following purification each individual majorly abundant extracellular product exhibited one band corresponding to its respective molecular weight when subjected to polyacrylamide gel electrophoresis, thereby allowing individual products or groups of products corresponding to the majorly abundant extracellular products to be identified and isolated. The purified majorly abundant extracellular products have been characterized and distinguished further by determining all or part of their respective amino acid sequences using techniques common in the art. Sequencing may also provide information regarding possible structural relationships between the majorly abundant extracellular products and those of other pathogens.
Although effective at providing a new class of vaccines operating on a new functional mechanism, the prior art has yet to effectively address the therapeutic treatment of infection by pathogenic intracellular organisms. This is particularly true for those pathogenic organisms that have developed resistance to conventional antibiotic drugs. Accordingly, it is a principal object of the present invention to provide a new class of prophylactic and chemotherapeutic agents and methods for their production and use in combatting infectious intracellular pathogens, including intracellular bacterial pathogens.
It is another object of this invention to provide prophylactic and chemotherapeutic methods and agents for treatment of diseases caused by intracellular mycobacterial pathogens including M. tuberculosis, M. bovis, M. avium, M. kansasii, M. fortuitum, M. chelonei, M. marinum, M. scrofulaceum, M. leprae, M. africanum, M. ulcerans and M. microti.
It is an additional object of this invention to provide such prophylactic and therapeutic methods and agents which exhibit reduced toxicity relative to other known treatment methods.
It is another object of this invention to provide antibiotics which are effective against diseases caused by drug-resistant strains of intracellular pathogens present in mammalian hosts.